The present invention relates to a new method of synthesising peptides containing one or more amino acid residues bearing an N-carbamoyl functional group. The invention also relates to new synthesis intermediates used in this method and to new peptide fragments.
Various synthetic peptides, used especially as reagents for analytical tests, as sweetening agents, as synthetic hormones or as medicinal products, contain amino acid residues bearing an N-carbamoyl functional group, either on the .alpha.-amino functional group of the N-terminal residue, or on the .omega.-amino functional group of a diamino acid residue, that is to say at the end of a side chain. Such diamino acids are especially norcitrulline (Nci), citrulline (Cit) or homocitrulline (Hci).
The introduction of an amino acid residue bearing an N-carbamoyl functional group into a peptide chain, starting directly with the amino acid carrying this carbamoyl functional group, has some disadvantages. Indeed, the presence of a ureido or ureino functional group seriously interferes with known methods of peptide synthesis, both in the "solid" phase based on the so-called Merrifield technique, and in the liquid phase. The chemical yields are poor and an adequate chiral purity is obtained only at the cost of laborious and costly purifications. Furthermore, in the case of diamino acids, the synthesis of amino acids bearing a ureido or ureino functional group at the end of the side chain, in pure enantiomorphous form, is difficult and therefore costly.
The synthesis of a peptide containing an amino acid residue bearing an N-carbamoyl functional group is therefore generally carried out in two stages. In a first step, a precursor peptide containing the amino acid residue bearing the amino functional group to be carbamoylated is synthesised. In a second step, the amino functional group to be carbamoylated is converted, inside the peptide chain, to a ureino functional group. For example, it is well known to convert, by carbamoylation by means of sodium isocyanate, the .alpha.-amino functional group of the N-terminal valine residue of the .beta. chain of sickle cell hemoglobin, to a ureido functional group. (Lehninger A. L.; Biochemistry; (1975) second Edition, p. 149)
However, this known method does not appear to be satisfactory for peptide synthesis, mainly because of the insufficient specificity of the cyanates or isocyanates for the amino functional group(s) to be carbamoylated and because of the racemisation which may be induced by such a treatment.
Moreover, R. A. Boissonnas and G. Preitner (Helvetica Chimica Acta, (1953), Vol. 36 (4), p. 875-886) studied the removal of phenyloxycarbonyl (PhOC) and p-tolyloxycarbonyl (TOC) groups, which are used as groups for blocking the .alpha.-amino functional group of some .alpha.-amino acids, by means of various dissociating agents. All the dissociating agents used led to the complete removal of these groups and to the release of the .alpha.-amino functional group.
The invention overcomes the disadvantages of known methods of preparing N-carbamoyl-peptides by providing a new method for preparing N-carbamoyl-peptides with an improved chemical yield, which makes it possible to preserve to a remarkable extent the chiral purity of the structures used.